Project Summary and Abstract Genetic information is stored as chromatin, a well-organized assembly of DNA and proteins that must be dynamically packed and unpacked to regulate gene expression. One way chromatin packing is modified is through exchange of canonical histone proteins in nucleosomes with histone variants, such as the replacement of histone H2A with histone H2A.Z. H2A.Z is a non-canonical histone variant that is highly conserved across all species. A special class of nucleosome remodelers insert H2A.Z into nucleosomes via an ATP-dependent process. Regulation of gene expression by insertion of H2A.Z into nucleosomes is directly related to a wide range of biological processes, such as the formation of long-term memory, as well as cellular dysfunction. H2A.Z overexpression is a common trait of breast, lung, colorectal and bladder cancers. Up- and down-regulation of the nucleosome remodelers SRCAP and p400, which are responsible for H2A.Z exchange in humans, are also associated with a number of cancers and diseases. Despite its importance, our molecular- level understanding of enzyme-mediated histone exchange is poor. In three specific aims, I propose to fill this knowledge gap by studying how the yeast nucleosome remodeler SWR1, a 14-subunit multiprotein complex, inserts H2A.Z-H2B dimers into nucleosomes. Aim one is to develop a single molecule assay where each step of SWR1-mediated histone exchange can be monitored in real time. Aim two will probe how the protein Yaf9, a subunit in the SWR1 complex, helps to couple the energy produced from ATP hydrolysis to the process of histone exchange. Aim three is to develop a single molecule assay that can distinguish between exchange of H2A.Z-H2B with H2A-H2B in nucleosomes that is proximal or distal to linker DNA, providing insight into why H2A.Z is enriched on the side of nucleosomes opposite to the nucleosome free region of promoters. The results of these experiments will significantly contribute to our fundamental understanding of chromatin biology and will aid efforts that are using the histone exchange process as a therapeutic target for cancer and long-term memory disorders.